1. Field of the Invention
The present invention relates to the absolute characterization of microscopic particles in solution. More particularly, the present invention relates to the absolute characterization of microscopic particles, such as polymers and colloids using static light scattering (SLS) and time-dependent static light scattering (TDSLS). In principle, the size range of detectability should run from about 20 Angstroms to 100 microns, with useful measurability in the range from 20 Angstroms to 2 microns, and a preferred range from about 20 Angstroms to 5000 Angstroms. Stated in terms of molar mass, the detectable range of particles should run from about 500 g/mole to 1014 g/mole, with useful measurability in the range of 500 g/mole to 109 g/mole, with a preferred range from about 1000 g/mole to 107 g/mole.
The preferred use of this invention is the determination of average particle masses, static dimensions, interaction coefficients, and other properties, as well as their changes in time, when scattering is from a very large number of particles. This is to be distinguished from turbidometric and nephelometric techniques, in which turbidity or relative scattering of solutions is measured and compared to relative reference solutions, in order to obtain concentrations of particles. The SLS technique employed refers to absolute macromolecular characterization, and not to determinations of concentrations of particulates with respect to specific relative calibrations, etc. This is also to be distinguished from devices which count and characterize single particles, although the present invention can count and characterize single particles, in addition to making SLS measurements. The least number of particles whose scattered light would be detected in the scattering volume (the volume of illuminated sample whose scattering is measured by a given photodetector) would be on the order of 20 and the maximum on the order of 4×1017, with the preferred range being from about 15,000 to 1.5×1013 particles. In terms of concentration of solute (dissolved polymer or colloid) the range would be from about 10−8 g/cm3 (for very large particles) to 0.2 g/cm3 (for very small particles) with the preferred range being from about 10−6 to 10−1 g/cm3. It should be pointed out that SLS in the absolute mode requires optically transparent solutions in which single, not multiple, scattering dominates. Many particle concentration detectors actually work in turbid solutions, which is a different range of conditions entirely.
SLS has proven to be a useful technique not only for characterizing equilibrium properties of microscopic particles, such as molar mass, dimensions and interactions, but also for following time-dependent processes such as polymerization, degradation and aggregation. Measuring the time-independent angular distribution and absolute intensity of scattered light in the equilibrium cases allows the former properties to be determined, according to procedures set forth by Lord Rayleigh, Debye, Zimm and others (e.g. ref. 1). In particular, this invention can be used in conjunction with the well known procedure of Zimm to determine weight average molar mass Mw, z-average mean square radius of gyration <S2>z and second virial coefficient A2. Measuring the time-dependent changes in the scattered intensity allows calculation of kinetic rate constants, as well as deduction of kinetic mechanisms and particle structural features (e.g. refs. 2,3). TDSLS can be used to monitor polymerization and degradation reactions, aggregation, gelling and phase separation phenomena (e.g. ref. 4).
In addition to absolute SLS and TDSLS measurements, the present invention can also simultaneously count and characterize individual particles which are much larger than the principal polymer or colloid particles; e.g., the large particles may have a radius of 5 microns, whereas the polymer may have an effective radius of 0.1 micron. The large particles may represent a contaminant or impurity, or may be an integral part of the solution, e.g., bacteria (large particles) produce a desired polymer (e.g., a polysaccharide) in a biotechnology reactor. The number density of bacteria can be followed in time, and the absolute macromolecular characterization of the polysaccharide could also be made (an auxiliary concentration detector would also be necessary if the polysaccharide concentration changes in time).
The present invention involves automatic online mixing and/or dilution of solutions containing polymers and/or colloids in order to provide relative and/or absolute characterization of these microscopic particles in solution. In the following, the term ‘dilution’ will be used, because, whenever two or more solutions are mixed, as described herein, the solutes in each will become dilute. The automatic dilution is intended to replace the traditional prior art of manually diluting such polymer/colloid solutions in order to make characterizing measurements, and to extend measurement capabilities to novel situations, especially those involving non-equilibrium (that is, time-dependent) processes, such as polymerization, degradation, aggregation and phase separation. The method can be used in conjunction with a variety of detectors, such as static light scattering (SLS), time-dependent static light scattering (TDSLS), heterogeneous time dependent light scattering (HTDSLS), dynamic light scattering, refractometry, ultraviolet and visible spectrophotometry, turbidometry, nephelometry, viscometry and evaporative light scattering. The automatic, online dilution of polymer and/or colloid solutions will be shown to have broad applicability in many sectors. In referring to the ensemble of SLS, TDSLS and HTDSLS detectors and methods in the following, the term light scattering (LS) will be used for brevity.
In principle, the size range of detectability of the polymers and/or colloids should run from about 20 Angstroms to 100 microns, with useful measurability in the range from 20 Angstroms to 20 microns, and a preferred range from about 20 Angstroms to 5000 Angstroms. Stated in terms of molar mass, the detectable range of particle molar masses should run from about 500 g/mole to 1014 g/mole, with useful measurability in the range of 500 g/mole to 1011 g/mole, with a preferred range from about 1000 g/mole to 1010 g/mole.
This invention focuses on automated methods that are used to characterize equilibrium and non-equilibrium properties of solutions containing polymers and/or colloid particles. Characterization of polymers and colloids via LS detectors is in terms of average particle masses, static dimensions, interaction coefficients, and other properties, as well as their changes in time, when scattering is from a very large number of particles. When large colloidal particles are present, the use of the method in conjunction with HTDSLS also allows the determination of the number density of these particles, information on their dimensions, and, when the system is not in equilibrium, how these properties change in time.
SLS has proven to be a useful technique for characterizing equilibrium properties of microscopic particles, such as molar mass, dimensions and interactions, and TDSLS and HTDSLS for following time-dependent processes such as polymerization, degradation and aggregation. Measuring the time-independent angular distribution and absolute intensity of scattered light in the equilibrium cases allows the former properties to be determined, according to procedures set forth by Lord Rayleigh, Debye, Zimm and others (e.g. ref. 1). In particular, this invention can be used in conjunction with the well known procedure of Zimm to determine weight average molar mass Mw, z-average mean square radius of gyration <S2>z and second virial coefficient A2. Measuring the time-dependent changes in the scattered intensity allows calculation of kinetic rate constants, as well as deduction of kinetic mechanisms and particle structural features (e.g. refs. 2,3). TDSLS can be used to monitor polymerization and degradation reactions, aggregation, gelling and phase separation phenomena (e.g. ref. 4).
In addition to absolute SLS and TDSLS measurements, use of the present invention in conjunction with HTDSLS allows simultaneous counting and characterization of individual particles which are much larger than the principal polymer or colloid particles; e.g., the large particles may have a radius of 5 microns, whereas the polymer may have an effective radius of 0.1 micron. The large particles may represent a contaminant or an impurity, or may be an integral part of the solution, e.g., bacteria (large particles) produce a desired polymer (e.g., a polysaccharide) in a biotechnology reactor. The number density of bacteria can be followed in time, and the absolute macromolecular characterization of the polysaccharide could also be made (an auxiliary concentration detector would also be useful if the polysaccharide concentration changes in time).
The method whereby simultaneous, absolute characterization of polymers and number counting of large particles is carried out, is described in U.S. patent application Ser. No. 08/969,386 (now U.S. Pat. No. 6,052,184). To optimize the technique, one should make the sample liquid flow relative to the irradiating laser beam (or other light source) in the scattering chamber, so as to produce countable scattering spikes as each large particle passes through the detected portion of the illuminated volume (the ‘scattering volume’), while ensuring, via correct design of the optical and electronic detection system, that there is on the average less than one large particle in the scattering volume at any given time. This allows the scattering level to recover to the baseline scattering of the pure polymer between the scattering spikes due to the large particles, so that the polymer can be absolutely characterized. The fraction of baseline time termed herein ‘clear window time’, and is detailed mathematically in ref. 5, wherein the method has recently been demonstrated. In this demonstration, it was first shown that useful characterization of a polymer solution could be made even in the presence of a large amount of particulate contamination. The contaminant was a known amount of 2 micron latex spheres introduced in increasing amounts to an aqueous polymer solution containing the polymer poly(vinyl pyrrolidone), or PVP. Secondly, the ability to simultaneously make absolute characterization of the polymer while the change in time of the large particle population was monitored was demonstrated by monitoring the growth of E. Coli bacteria amidst an aqueous solution of PVP polymer.
The present invention also involves the automatic extraction and dilution of high viscosity fluids.
More particularly, the present invention also includes a device for automatically and continuously sampling and diluting liquids of high viscosity, normally containing synthetic and/or biological polymers, to such an extent that absolute light scattering and/or other optical and physical measurements can be made. In many cases the viscosity in the vessel containing the fluid will vary continuously from a low value to a high value (polymerization reaction), or vice versa (degradation or phase separation reaction). In some instances it will be desirable to manually or automatically change the dilution factor during the course of a reaction or monitoring process.
2. General Background of the Invention
There is currently considerable interest in the polymer industry for finding a means of monitoring and controlling, in real-time or near real-time, the progress of polymerization and other reactions. Here, ‘polymer industry’ is understood to mean all industries producing synthetic polymers (e.g. polyolefins), as well as those producing or modifying biological or bioactive polymers, whether for food, pharmaceutical, cosmetic, or other applications. ‘Polymer reaction’ is understood to mean polymerization, copolymerization, degradation, or any means of modifying the chemical or physical properties of polymers.
Currently, the state of the polymer reaction can be found by manually sampling the reactor and making any number of analytical tests on the contents. This, however, leads to long delay times in obtaining results, usually too long to make useful adjustments to the reaction. Often times the analytical laboratory facilities are located remotely from the reactor. Such manual sampling also does not yield a continuous enough record of the reaction to follow the time course quantitatively. There can also be safety issues involved when workers expose themselves to hazardous reactor environments to obtain samples.
A step towards automation has been proposed recently by Symyx Technologies, Inc. (Ca.) and others, wherein a discrete, automatic sampling of reactor contents occurs, followed by injection of a finite volume of the extracted material into an analytical system, which contains a series of detectors, and, optionally, a chromatographic column to perform some separation of the injected material. This type of procedure leads to signal peaks in the detectors each time a sample is injected. The peaks are then normally analyzed using standard analytical practice to obtain molecular masses, degree of monomer conversion, and, sometimes, reduced viscosity. The actual sampling and dilution is normally carried out by a robotic system. For example, Waters introduced such an auto sampling system. All these techniques involve injection of a material to produce peaks, and yield data points separated by significant dead-times, during which the sampling and detector system recover in preparation for the next injected pulse of material. These techniques, including the manual one, can be termed ‘discrete sampling’ techniques.
The current invention builds off of an alternative sampling and analysis method, previously introduced by this inventor. This method is a continuous one, and does not involve injecting pulses of material and subsequently obtaining detector peaks for analysis. Recently, the inventor has coined the term Automatic, Continuous, Online Monitoring of Polymerization Reactions (ACOMP) for this method. In ACOMP a stream of material from the reactor is continuously mixed with a solvent, and the diluted mixture flows through the detector train, providing a continuous record of the reaction. In ACOMP no chromatographic columns are used, finite pulses of material are not injected into the detector train (although they may be injected into the mixing chamber), and detector signal peaks are not obtained. Another area of reaction monitoring involves in situ probes, such as near Infra-red and rheometers. While these probes allow real-time or near real-time data on the reaction to be gathered, they are inevitably empirical methods, largely based on chemometric approaches, which show a statistical relationship between a desired polymer property and an instrument's signal. ACOMP, in contrast, involves absolute measurements of molecular properties. ACOMP theory, practice and instrumentation, and related techniques, have been extensively described by the inventor and his co-workers. (Refs. 5, 6, 7, 8-17).
The single greatest problem in the practical use of ACOMP is the automatic, continuous preparation of the mixed or diluted sample which continuously feeds the detector train. The problem is due chiefly to the high viscosities which develop during many polymerization reactions, as well as the bubbles that can occur. Commercially available mixers are available, that use either high pressure (e.g. Dionex, Waters) or low pressure mixing schemes (e.g. Isco). The problem with these devices is that they are designed and built to handle only low viscosity liquids. When one of the feeds to a low pressure mixing pump is a reactor whose viscosity increases during a polymerization reaction, the mixing pump is incapable of maintaining a fixed volume withdrawal rate percentage. The result is that the lag time between withdrawal from the reactor and arrival of the mixed solution at the detectors becomes longer and longer as the reaction proceeds, often times to unacceptable levels. When a high pressure mixing scheme is used, bubbles produced either by the reaction itself, or due to cavitation during pump withdrawal, lead to the depriming of the withdrawal pump, and failure to continue monitoring. The check valves and other plumbing in such pumps is also susceptible to becoming frozen by plugs of polymeric material that can solidify in the pumps during operation. It is apparent that pumps that rely on pulling reactor material with a vacuum (1 atmosphere or less) are wholly unsuitable for ACOMP when viscosities are above about 150 centipoise (cP); i.e. arrangements of such pumps can typically follow a reaction from about 1 cP to about 150 cP.
On the other hand, a variety of pumps exist that can handle highly viscous materials. Certain peristaltic pumps, for example can pump liquids up to tens of thousands of cP, whereas gear, lobe and screw pumps can move liquids of millions of cP. Whereas this latter technology is highly developed for industries involving, for example, plastic injection and synthetic fiber production, there is no available system that can accomplish the prerequisite of ACOMP: Continuously withdraw a very small flow rate of material and mix it homogeneously with a solvent.
More information about the background of the inventions disclosed and claimed herein can be found in my patent applications mentioned herein.
Incorporated by reference are the following papers: Florenzano, Strelitzki and Reed, Macromolecules, vol. 31, pp. 7226-7238, 1998, “Absolute, On-line Monitoring of Molar Mass during Polymerization Reactions”; Strelitzki and Reed, Journal of Applied Polymer Science, vol. 73, pp. 2359-2368, 1999, “Automated Batch Characterization of Polymer Solutions by Static Light Scattering and Viscometry”; Schimanowski, Strelitzki, Mullin, and Reed, “Heterogeneous Time Dependent Static Light Scattering”, Macromolecules, (copy attached to U.S. patent application Ser. No. 09/404,484).